1. Field of the Invention
This invention relates to a process for constructing a recombinant strain of microorganisms which has desirable metabolic characteristics. In one of its more particular aspects, this invention relates to a multiple continuous culture method for producing recombinant bacteria. In another of its more particular aspects, this invention relates to the construction of a strain of microorganisms which is useful in the disposal of toxic wastes such as halogenated organic compounds.
Halogenated organic compounds, such as chlorobenzenes, are used in a wide variety of synthetic and utilitarian applications in industry, agriculture and health care. For example, halogenated organic compounds are used in dielectric fluids, flame retardants, refrigerants, heat transfer fluids, protective coatings, pesticides and other chemical products. Disposal of these materials after use in halogenated by-products of their production poses a serious problem because of the toxicity of these halogenated organic compounds. The disposal of toxic waste has, in recent years, achieved such huge proportions that storage of toxic chemicals in landfills and other areas designated for storage thereof no longer satisfies the ever growing need for safe and efficient disposal of these materials.
2. Prior Art
Many different methods have been proposed for rendering toxic waste innocuous. Among these are incineration, chemical transformation and microbiological degradation. Because microbiological degradation of toxic waste does not involve the use of chemical reagents which might themselves be toxic and does not result in the production of large amounts of noxious fumes, such as produced in the incineration of toxic waste, it has become a preferred method of disposing of toxic waste.
Most microbiological degradations of toxic materials are based upon discovering a particular microorganism which will metabolize a toxic material, converting it to innocuous metabolic products, usually, in the case of organic compounds, carbon dioxide, water and salts. Finding microorganisms which can efficiently and safely convert toxic wastes into innocuous metabolic products is a highly complex procedure involving many arduous steps and requiring a significant expenditure of time.
One such procedure is taught in U.S. Pat. No. 4,493,895, wherein is described a process for microbial degradation of obnoxious organic wastes into innocuous materials. This process comprises the steps of (1) collecting a sample of material from the site contaminated with obnoxious chemicals; (2) enriching the microorganisms found living in the sample; (3) separating the strains of microorganisms capable of having different metabolisms for the various chemicals in the sample from the site, from each other; (4) purifying the strains which are capable of biodegrading the chemicals to be disposed of; (5) applying the strain to the locales of the contaminants to be disposed of; and (6) monitoring of removal of the contaminants at the locales of the contamination. It can be seen that this is indeed an involved procedure requiring large amounts of time and effort.
Another approach taught in U.S. Pat. No. 5,511,657, involves a process of treating chemical waste landfill leachates with activated sludge containing bacteria capable of metabolizing obnoxious organics present in the leachate.
U.S. Pat. No. 4,664,805, assigned to the same assignee as the present invention, describes a process in which environments contaminated with toxic halogenated organic compounds are decontaminated at an accelerated rate by the addition of (1) microorganisms which are non-indigenous to the environment in which it metabolizes a contaminant at a greater rate than microorganisms indigenous to the environment; and (2) a non-toxic analog of the halogenated organic compound.
The foregoing references describe processes in which reliance is placed upon naturally occurring microorganisms. Another approach involves the production of microorganisms having the same desired metabolic properties by genetic engineering or various related procedures. An example of such method is shown in U.S. Pat. No. 4,535,061 wherein plasmid-assisted molecular breeding procedures for generating pure and mixed cultures of microorganisms capable of dissimilating environmentally persistent chemical compounds are described.
Microorganisms which utilize chlorinated aromatic hydrocarbons as sole carbon sources cannot normally be isolated by conventional enrichment culture techniques. Nevertheless, many aromatic hydrocarbon-utilizing bacteria can co-metabolize these compounds. The dead end products formed from co-metabolism in pure culture may be further metabolized to CO.sub.2, HCl and H.sub.2 O by other microbial species as noted in the mineralization of PCB's in soil as described in W. Brunner, F. H. Sutherland and D. D. Focht, Enhanced Biodegradation of Polychlorinated Biphenyls In Soil by Analog Enrichment and Bacterial Inoculation, J. Environ. Qual. 14:324-328, 1985. Similar processes are described in D. D. Focht and W. Brunner, Kinetics of Biphenyl and Poly-chlorinated Biphenyl Metabolism in Soil, Appl. Environ. Microbiol. 50:1058-1063, 1985.
The mobilization of the requisite gene pool into a single species would be a desirable goal for complete mineralization of a target compound. Recombinants are obtained by genetic engineering techniques or by natural genetic exchange between bacteria. The former method is very tedious and time consuming, and is frequently limited by the lack of commercially available degradation products for isolation of clones specific for given catabolic functions. Natural genetic exchange, on the other hand, involves very little labor, but requires considerable patience and luck since the requisite organisms may either not exist or be present in such low numbers that the frequency of the desired genetic exchange event may be too low to be manifested over the observation period.
For example, enrichment cultures of Alcaligenes sp. that grew on 1,4-dichlorobenzene (1,4-DCB) required at least ten months for isolation as described in G. Schraa, M. L. Boone, M. S. M. Jetten, A. R. van Neervaen, P. J. Colberg and A. J. B. Zehnder, Degradation of 1,4-dichlorobenzene by Alcaligenes sp. strain A175, Appl. Environ., Microbiol. 52:1374-1381, 1986.
Similarly, cultures growing on 1,3-dichlorobenzene (1,3-DCB) required at least six months for isolation as described in J. A. M. DeBont, M. J. A. W. Vorage, S. Hartmans and W. J. J. van den Tweel, Microbial Degradation of 13 Dichlorobenzene, Appl. Environ. Microbiol. 52:677-680, 1986.
Nine months of continuous selection pressure in a chemostat was required before growth on chlorobenzene (CB) with strain WR 1306, which was originally isolated from enrichment with benzene as described in W. Reineke and H.-J. Knackmuss, Microbial Metabolism of Haloaromatics: Isolation and Properties of a Chlorobenze-Degrading Bacterium, Appl. Environ. Microbiol. 47:395-402, 1984.
If two or more organisms together, but not separately, possess the catabolic enzymes for complete mineralization of a substrate, then it should theoretically be possible to construct a recombinant strain that would grow with that substrate as its sole carbon source if selection pressures can be optimized separately for the recombinant and the parental strains.
It is accordingly an object of the present invention to provide a process for the construction of a recombinant strain of microorganism which possesses the combined catabolic enzymes present in the parent bacteria. Another object of this invention is to provide a process for constructing recombinants which proceeds at a faster rate than previously available processes. Other objects and advantages of the present invention will become apparent in the course of the following detailed description.